Fig 1: H3.3 incorporation into chromatin in response to metastatic signalling drives NADK expression in breast cancer. a, H3.3 enrichment at the NADK promoter in MCF-10A cells with CHAF1B suppression for 3 days; fold enrichment was determined using immunoglobulin G (IgG) as a control for the ChIP (n = 3). c, NADK immunoblot in HCC1806 cells with CHAF1B suppression for 3 days; representative images (n = 4). c, d, NADP+(c) and NADPH (d) levels in HCC1806 cells with CHAF1B knockdown for 3 days (n = 4). e, NADK immunoblot in HCC1806 cells with HIRA knockdown treated with TGFß + TNFa for 5 days; representative image (n = 4). f, g, NADP+(f) and NADPH (g) levels in HCC1806 cells with HIRA knockdown treated with TGFß + TNFa for 5 days (n = 4). h, H3.3 enrichment at the NADK promoter in LM2 cells with HIRA knockdown for 3 days; fold enrichment was determined using immunoglobulin G (IgG) as a control for the ChIP (n = 3). i, NADK mRNA quantity in LM2 cells with HIRA knockdown for 3 days (n = 4). j, NADK immunoblot in LM2 cells with HIRA knockdown for 3 days; representative image (n = 4). k, l, NADP+(k) and NADPH (l) levels in LM2 cells with HIRA knockdown for 3 days (n = 4). All values are expressed as mean ± SEM.
Fig 2: Depletion of HIRA, a positive control for the imaging-based screen, shows the mislocalization of CENP-A and CIN phenotypes in HeLaYFP–CENP-A-low cells. (A) Western blots of lysates prepared from HeLaYFP–CENP-A-low cells transfected with the indicated siRNAs for 72 h and analyzed using the antibodies as indicated. (B) Representative images of metaphase chromosome spreads showing the localization of CENP-A at centromeric and non-centromeric regions in HeLaYFP–CENP-A-low cells transfected with the indicated siRNAs. Metaphase chromosome spreads were prepared 72 h post transfection, and cells were immunostained using an antibody against CENP-A and stained with DAPI. Scale bars: 5 µm. (C) Quantification of CENP-A signal intensities at centromeric (left) and non-centromeric (right) regions in metaphase chromosome spreads of HeLaYFP–CENP-A-low cells transfected with the indicated siRNAs. Each circle represents a spot on a centromeric or non-centromeric region. n denotes the number of chromosomes analyzed. Red lines indicated mean±s.e.m. for YFP signal intensities across areas measured in the number of spots indicated as n from three independent experiments. A.U., arbitrary units. (D,E) Immunostained images (D) and bar chart (E) showing the proportion of cells with defective chromosome segregation in HeLaYFP–CENP-A-low cells transfected with the indicated siRNAs. The yellow arrow shows lagging chromosomes. Error bars represent s.e.m. across three independent experiments. n denotes the number of cells analyzed from three experiments. P-values shown in C, E were calculated using unpaired, two-tailed t-test.
Supplier Page from Abcam for Anti-HIRA/HIR antibody [EPR7416]